CD45.1 or Thy1.1 congenic OT-1 TCR transgenic naïve T cells were isolated using the CD8 untouched isolation kit (Miltenyi Biotech) plus biotin conjugated anti-CD44. Cells were transferred into C57/BL6 (Jackson Laboratories) mice 1 day prior to infection.
Recombinant Listeria monocytogenes strains were generated that stably express chicken Ovalbumin (AA134–387) containing either the native ligand SIINFEKL257–264 or the APL listed inSupp. Fig. 2 . The designation of the APL indicates the substituted amino acid and the position within the SIINFEKL epitope. A previously described cassette24 (link) encoding for the expression of secreted Ova was manipulated by site-directed mutagenesis to insert the APL. The cassettes were cloned into a vector (pPL2 ) and stable Listeria recombinants were made as described25 (link). Mice were infected i.v. with 1000 CFU of log phase Listeria. For VSV-N4ova, mice were infected i.v. with 2×105 PFU.
Spleens were digested with Blendzyme 2 and DNAse 1 (both Roche) for 1 hour at 37°C and mashed through a 100 µm cell strainer (Becton Dickinson) to obtain single cell suspensions. Flow cytometry, intracellular cytokine staining (ICS), and CFSE labeling were performed using standard procedures. For ICS, the cells were first stimulated at 37°C for 30 min with peptide, then 7 µM Brefeldin was added and the incubation was continued for another 4.5 hours. For CCR7 staining, cells were incubated for 1 hour at 37°C and then stained on ice with CCL19-IgG fusion supernatant26 (link). To assess the level of non-specific binding of CCL19-IgG to OT-1, soluble CCL19 (R&D Systems) was added to control samples.
For immunofluorescent microscopy, spleens were fixed in 4% paraformaldehyde and kept in 30% sucrose over night. 10–20 µM sections, were cut and stained using standard procedures and analysed on a Zeiss 510 META confocal microscope.
Recombinant Listeria monocytogenes strains were generated that stably express chicken Ovalbumin (AA134–387) containing either the native ligand SIINFEKL257–264 or the APL listed in
Spleens were digested with Blendzyme 2 and DNAse 1 (both Roche) for 1 hour at 37°C and mashed through a 100 µm cell strainer (Becton Dickinson) to obtain single cell suspensions. Flow cytometry, intracellular cytokine staining (ICS), and CFSE labeling were performed using standard procedures. For ICS, the cells were first stimulated at 37°C for 30 min with peptide, then 7 µM Brefeldin was added and the incubation was continued for another 4.5 hours. For CCR7 staining, cells were incubated for 1 hour at 37°C and then stained on ice with CCL19-IgG fusion supernatant26 (link). To assess the level of non-specific binding of CCL19-IgG to OT-1, soluble CCL19 (R&D Systems) was added to control samples.
For immunofluorescent microscopy, spleens were fixed in 4% paraformaldehyde and kept in 30% sucrose over night. 10–20 µM sections, were cut and stained using standard procedures and analysed on a Zeiss 510 META confocal microscope.